JOUIRNAL OF BACTERIOLOGY, May 1978, P. 440-445 0021-9193/78/0134-0440$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 134, No. 2 Printed in U.S.A.

Early-Blocked Asporogenous Mutants of Bacillus subtilis Are Lysogenized at Reduced Frequency by Temperate Bacteriophages TOSHIHIKO IKEUCHI* AND KIYOSHI KURAHASHI

Institute for Protein Research, Osaka University, Yamada-kami, Suita, Osaka 565, Japan Received for publication 5 January 1978

The establishment of lysogeny in early-blocked asporogenous (Spo-) mutants of Bacillus subtilis 168, which were also defective in the production of antibiotics (Abs-), by temperate phage 4105 or SPO2 was studied. It was found that the frequency of lysogenization of Spo- Abs- mutants was 10 to 20% that of the wildtype bacteria. There was no difference in the efficiency of plating and the burst size of 4105 between the wild-type and mutant strains. 4105 lysogens of mutant strains were as stable as those of the wild type. Several rifampin-resistant mutants defective in the production of antibiotics were isolated. They were also defective in spore formation and lysogenized by 4105 at reduced frequency. When Bacillus subtilis is cultured in sporulation media, the initiation of sporulation occurs at the end of the vegetative phase of growth. Processes which follow result in the formation of endospores within several hours (9, 27, 30). The production of antibiotics is one of the early sporulation events (22, 30), but the structure and function of the antibiotics of B. subtilis 168 are unknown. Losick and his collaborators reported that the activity of the sigma subunit of B. subtilis RNA polymerase (EC 2.7.7.6) decreased during the first hours of sporulation (20, 21, 35, 40) and that the sigma subunit of some mutants blocked in sporulation remained active during the stationary phase (5, 34, 35, 40). However, the relationship between the loss of the sigma activity and the early sporulation events, especially the production of antibiotics, is not clear. The initiation of sporulation of B. subtilis cells is considered to occur at the end of vegetative growth by recognizing the change of their microenvironment. If the early-blocked asporogenous (Spo-) mutants were defective in this recognition process, these mutants might be pleiotropically defective in other functions occurring during, and at the end of, the vegetative phase of growth. A number of workers have investigated the differences in the vegetative phase of growth between the wild-type and early-blocked asporogenous mutant bacteria (3, 4, 16, 19). In this report, we describe another difference, concerned with temperate phage growth in vegetative cells, between the wild type and Spo- mutants. Virulent phage infection has been used to investigate the control mechanisms associated

with sporulation (16, 36, 39, 41). Recently, Osburne and Sonenshein reported (25) the behavior of a temperate phage (4i105) in sporulating cells of B. subtilis. Our studies are concemed with the behavior of two temperate phages, 4105 (2) and SPO2 (24, 28), in the vegetative cells of asporogenous mutant strains of B. subtilis. These two phages are similar in their physical properties and behavior in the infected cells (2, 24), but their attachment sites (att) are different (14, 29; see Fig. 1). MATERIALS AND METHODS Bacterial strains. The strains of B. subtilis used in this study are listed in Table 1. Staphylococcus aureus ATCC 4012 was used as an indicator strain for the detection of antibiotic production by B. subtilis. Media. LB medium contained 10 g of tryptone (Difco), 5 g of yeast extract (Difco), 1 g of glucose, and 5 g of NaCl per liter; the pH was adjusted to 7.1 before sterilization. NB medium contained 16 g of nutrient broth (Difco) and 2.5 g of NaCl per liter. Schaeffer medium (31) contained 8 g of nutrient broth (Difco), 0.25 g of MgSO4 7H20, and 1 g of KCI per liter, 1 mM Ca(NO3)2, 0.1 mM MnCl2, and 1 uM FeSO4. The sporulation medium (2x Schaeffer medium) was essentially the same as the medium of Schaeffer as modified by Korch and Doi (17). It consisted of 16 g of nutrient broth (Difco) per liter, 1 mM MgSO4, 13 mM KCI, 1 mM Ca(NO3)2, 0.1 mM MnCl2, 1 ,uM FeSO4, and 5 mM glucose. Spizizen minimal medium (37) contained 14 g of K2HPO4, 6 g of KH2PO4, 2 g of (NH4)2SO4, and 1 g of sodium citrate per liter, 1 mM MgSO4, and 25 mM glucose. Solid media (hard agar) usually contained 1.5% agar, and soft agar contained 0.7% agar. Preparation of phage lysates. Bacteriophage c0105 and SPO2 lysates were prepared by induction of lysogens with mitomycin C. Lysogens 168(q,105) and

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TABLE 1. Bacterial strains Genotype Phenotypea Origin/source trpC2 Spo+ S. Okubo trpC2 spoA12 SpoA J. Spizizen (11) spoOB6Z SpoOB P. Schaeffer (15) trpC2 spoOC9V SpoOC P. Schaeffer (15) spoIII94U SpoIII P. Schaeffer (15) spoIVIIT SpoIV P. Schaeffer (15) SR22Revlb trpC2 Spo+ Abs+ SR22 9VReVlb trpC2 Spo+ Abs+ 9V 9VRev2b trpC2 Spo+ Abs+ 9V 4455 trpC2 spo-4 rif-4 Spo- Abs- Cpt168 5221 trpC2 spo-5 rif-5 Spo- Abs- Cpt168 C14 cysA Spo+ S. Okubo 168(4105) trpC2 (4105) Spo+ H. Saito 168(SPO2) trpC2 (SPO2) Spo+ H. Saito Spo, Sporulation; Abs, production of antibiotics; Cpt, competence for genetic transformation. b Spontaneous Spo+ revertants were isolated from the survivors of strain SR22 and 9V cells grown overnight in Schaeffer medium and heated at 800C for 10 min. Strain 168 SR22 6Z 9V 94UL llT

168(SPO2) were grown in LB medium to 1 x 108 to 2 x 108 cells per ml and treated with mitomycin C (1 Ag/ml) at 37°C for 10 min. The cells were centrifuged and suspended in LB medium containing 5 mM MgSO4, 3 mM CaCl2, and 0.01 mM MnCl2. After the cultures were aerated for 2 h, the lysates were treated with CHC13 and cleared by centrifugation. Lytic growth lysates of 4105 and SP02 phages were obtained from cells of strain 168 grown in LB medium containing 5 mM MgSO4, 3 mM CaC12, and 0.01 mM MnCl2, infected with phages at a multiplicity of about 5, and aerated for 1 h. The titer of phage 0105 or SP02 lysates was about 2 x 1010/ml. Frequency of lysogenization. A bacterial culture in 5 ml of sporulation medium was grown to a turbidity of about 100 Klett units (no. 54 filter), and a 0.5-ml inoculum from this culture (in exponential growth) was transferred to 5 ml of sporulation medium twice in succession; 0.25 ml of the final culture grown to a turbidity of 50 Klett units (ca. 5 x 107 cells per ml) was mixed with 0.02 ml of a 4105 or SP02 lysate diluted with sporulation medium at a multiplicity of infection of about 20. After treatment at 370C for 15 min, the culture was diluted with Spizizen minimal medium (or sporulation medium) and plated on Schaeffer medium. Plates were incubated at 370C for 1 day. The number of lysogens was determined by a cross-streak test for immunity of the surviving colonies to phage. The total number of cells was determined similarly by plating the uninfected culture on hard agar after dilution. Under this condition, the number of uninfected cells was less than 1% of the total, and the number of infective centers (number of infected cells) measured after removal of free phages by centrifugation was nearly the same as the total. Efficiency of plating. Bacterial cultures grown to the exponential phase (turbidity of 50 Klett units), as for the determination of lysogenization frequency, were mixed with 4105 lysates diluted with sporulation medium. The phage titer was determined by using a soft-agar overlay of LB medium on an LB plate. Efficiency of plating was defined as the ability of a mutant strain to form plaques with phage 4105 relative to that of the wild-type strain.

Burst size. A bacterial culture of a mutant strain grown to a turbidity of 50 Klett units was mixed with phage 0105 at a multiplicity of infection of 0.2. After 10 min of adsorption at 370C, the culture was diluted 10,000-fold with sporulation medium and shaken at 370C. The number of infective centers immediately after dilution was determined by using a soft-agar overlay of LB medium, from which CaC12, MgSO4, and MnCl2 were omitted, containing a culture of the same strain. The phage titer after incubation at 370C for 60 min was determined by using a soft-agar overlay of LB medium after treatment of a culture of the same strain in LB medium containing 3 mM CaC12, 5 mM MgSO4, and 0.01 mM MnCl2. The average burst size was the ratio of these two values. Spore-forming ability. Bacterial cells in Schaeffer medium were grown at 370C for at least 12 h beyond the end of the vegetative phase of growth, and the number of viable cells was determined by diluting the cells and plating them on hard-agar Schaeffer medium. The number of spores was determined by plating the cells on hard agar after heating the culture at 800C for 10 min. Production of antibiotics. The ability to produce antibiotics was determined by the procedure of Spizizen (38) with some modifications. A suspension of cells to be tested was spotted on soft-agar Schaeffer medium containing S. aureus (31). The plates were incubated at 370C for 1 day. Abs+ denotes the formation of a clear inhibition zone on the lawn of the indicator bacteria. Isolation of rifampin-resistant mutants defective in the production of antibiotics. Rifampin is known to be a specific inhibitor of RNA polymerase. Rifampin-resistant (Rif') mutants were selected from B. subtilis 168. A logarithmic-phase culture (1 x 10' to 2 x 10' cells per ml) of strain 168 was treated with 50 jig of N-methyl-N'-nitro-N-nitrosoguanidine per ml for 30 min. Survival of the cells under this condition of mutagenesis was 10 to 20%. The cells were centrifuged, washed twice with LB medium, resuspended in LB medium, and shaken overnight at 370C. The overnight culture was diluted and plated on NB agar containing 20 ug of rifampin per ml. The frequency of mutation

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to rifampin resistance was 10'. About 5,000 rifampinresistant mutants were tested for the production of antibiotics by the procedure described above. Five independent mutants defective in the production of antibiotics (Abs- or Abs') were isolated. All of these were simultaneously defective in spore formation. Two Rif' Abs- Spo- mutants (4455 and 5221) were used in this investigation. Transduction with phage PBS1. Phage PBS1 was obtained from H. Saito of Tokyo University. The transduction experiments were carried out essentially by the methods of Lennox (18), except that phage PBS1 and B. subtilis strains were employed. Transformation. Transformation experiments were carried out by the procedure of Anagnostopoulos and Spizizen (1) as modified by Okubo (23). Chemicals. Rifampin was obtained from Lepetit (Milan), and streptolydigin (U-5481) was kindly donated by G. B. Whitfield, Jr., the Upjohn Co., Kalamazoo, Mich.

RESULTS

Frequency of lysogenization. 0105 forms turbid plaques on B. subtilis 168 because it is a temperate phage (2). However, when an earlyblocked asporogenous mutant, SR22 or 6Z (Table 1), of B. subtilis 168 was infected with 4)105, the plaques were clearer than those on the wildtype cells. Quantitative determination of lysogenization frequency of several strains of mutant bacteria by temperate phages 4105 and SPO2 was thus carried out (Table 2). With the earlyblocked Spo- mutants SR22 and 6Z (both SpoAbs-), the frequency of lysogenization by 4105 or SPO2 was reduced to 10 to 20% that of the wild type, whereas with mutants 94UL (SpoIII) and lIT (SpolV) the frequency was similar to that of the wild-type bacteria. Both 4105 and SPO2 gave similar results, although SPO2 showed a higher frequency of lysogenization in the wild-type strain. Lysogenization frequencies of mutant 9V by both 4105 and SPO2 were found to be between TABLE 2. Frequency of lysogenizationa Phage +105 Strain

Frequency

of lysogenizationh

Phage SP02

Frequency

Ratio

(%)

of lysogenizationh

Ratio

(%)

100 11

11 100 1.6 15 14 0.71 6.5 27 3.6 33 107 10 91 llT 93 10 91 a Induced phage lysates were used. The same values were obtained with lytic growth lysates. b Frequency of lysogenization among infected cells [(lysogens/original number of cells) x 100] was measured as described in the text.

168 SR22 6Z 9V 94UL

4.1 0.45 0.56 1.1 4.4 3.8

those of the wild-type strain and Spo- Absmutants. Mutant 9V was classified as SpoOC and Abs+ by Scheaffer (15, 30), but in our hands it was found to be Spo- Abs', suggesting that the block of the early sporulation events is leaky. With one Spo+ Abs+ revertant of mutant SR22 (SR22Revl in Table 1), the lysogenization frequency by 0105 recovered to the same level as that of the wild type (4.1%). With 9VRevl, the frequency was 3.6% (about 90% compared with that of the wild type), and with 9VRev2, another independent isolate, it was 2.9% (about 71%). The growth rates of the five Spo- mutants mentioned above were the same as that of the wild type. Therefore, this phenomenon of reduced lysogeny is not due to the secondary effect of the lowered growth rate of the host strains. Growth of phage 4105 in Spo- mutants. Table 3 shows the efficiency of plating of 4)105 and the average burst size in the vegetative cells. With Spo- mutant strains, the efficiency of plating of 4)105 was a little higher than that with the wild type, and the burst size in the lytic growth cycle was about the same as that with the wild type (2). These results indicate that adsorption and lytic growth of 4)105 in these Spo- mutants are normal. Maintenance of lysogeny. The effect of the spo mutations appeared to be on the establishment of lysogeny rather than on the maintenance of lysogeny. Although the frequency of lysogenization was low, it was possible to obtain lysogens from all of the mutant strains. These 4)105 lysogens were stable on repeated purification, and the titer offree phage in these lysogenic cultures was as low as in the culture of the lysogen of the wild-type strain (Table 4). That strain 168(4)105) is stable is consistent with the results reported by Birdsell et al. (2) and Osburne and Sonenshein (25). Isolation and properties of rifampin-resistant mutants defective in sporulation and the production of antibiotics. The mutation sites of all the above Spo- mutants are clustered in the pheA-lys region (11, 15; Fig. 1). TABLE 3. Growth of bacteriophage 4105' Strain

EOP

Burst size

1.0 254 168 1.4 271 SR22 1.6 6Z 264 262 9V 1.3 a The efficiency of plating (EOP) and burst size of 4105 on the cells in the vegetative phase of growth were determined as described in the text. Values are averages of duplicates. The differences between the values for the four strains are not considered to be significant. The burst sizes after UV induction of 4105 lysogens of these strains were 45 to 55.

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TABLE 4. Free phage in the cultures of )105

as to produce antibiotics as described in Materials and Methods. Five to ten percent of the Rif' mutants were Spo- Abs+, and 0.1% (five no.' Phage tiPhg Strain independent (xle) isolates) were Spo- Abs- (Spotiter/cell no. (x107) Abs'). Two of the Spo- Abs- mutants, 5221 and 2.7 168(4105) 5.8 4455, were studied further. 2.1 x 10-4 SR22(,105) 7.3 7.8 1.1 X 10-4 The properties of these two mutants are pre6Z(4.105) 4.6 2.3 5.0 x 10-6 sented in Table 5. The spore-forming abilities of 4.4 9.5 2.2 X 10-4 9V(W105) these mutants were lower than 2 x 10-4 that of ' Bacterial cultures were grown to the exponential the wild type (Osp), and the mutants were Cptphase (turbidity, 40 to 60 Klett units) as in the deter- (competence for genetic transformation). The mination of lysogenization frequency. The cell number frequency of lysogenization of these strains by was determined by plating the cells on hard-agar 4105 was found to be as low as that of the other Schaeffer medium after dilution with Spizizen minimal Spo- Abs- mutants described above. These remedium. b The same bacterial cultures were treated with sults suggest that the frequency of lysogenizaCHCL, at 370C for 10 min, and the cells were removed tion of all early-blocked asporogenous mutants by centrifugation (1,600 x g) for 10 min. After the may be low irrespective of the map positions of supernatant was incubated at 370C for 30 min to their mutation sites. Mapping of the 8po mutation in strain remove CHCl1, the phage titer was determined by using a soft-agar overlay of LB medium. 5221. Genetic analyses by transduction and transformation of one of the Spo- Abs- mutants, 5221, were carried out. Probably because of the treatment with nitrosoguanidine and the method of selection, this mutant strain had two mutations, rif and spo-5 (Table 6; Fig. 1). The cotransduction frequency and the cotransformation frequency of spo-5 with rif were 95 and 73%, respectively (Table 6). The frequency of lysogenization of a Spo- Abs- Rif' transformant by 0105 was also found to be low; that of a Spo+ Abs+ Rif' transformant was found to be high (data not presented). Transformation experiments of a spontaneously isolated streptolydigin-resistant (Stdr) mutant of 5221 were carried out. Of 100 Cys+ transformants, 41 Spo- transformants were Std', indicating that spo-5 was closely linked to an std site. At present, the relationship between the spo-5 gene and ts genes in this region, reported ocf by Haworth and Brown (8), is not clear. lys rif (std) cysA I I~ I ~~~~~~~~~~~~~~~~~~ Mutant 4455 was considered to have a mutaOe6z WIIT / / 1pOA12 spo-4 spo-5

lysogens

Cell

.

194U

OC9V

FIG. 1. Chromosomal map of B. subtilis. The map positions were taken from the map of Young and Wilson (42) and references 11 and 15. The map position of spo-5 (carried by strain 5221) was determined from the transformation experiment (three-factor crosses) of Tabk 6. Map distances between the two linear map loci cannot be compared.

To confirm that the findings concerning phage growth and lysogeny prevailed in Spo- mutants for which mutation sites were mapped in other loci, we isolated and characterized some rifampin-resistant Spo- Abs- mutants. About 5,000 rifampin-resistant mutants were selected and tested for the ability to sporulate on plates of hard-agar Schaeffer medium as well

TABLE 5. Properties of Rif Spo- Abs- mutants Lysogenization by 40105 Strain

Abs- Cptb

Spores/ cell

Frequency

Ratio

(%) 168 + + 1.0 4.1 100 4455 - 6.6 x 10-6 0.84 20 5221 - 1.9 x 10-4 0.44 11 " As described in the text. b Competence for genetic transformation (Cpt) was determined as follows. The cultures of mutants and control 168 were mixed with DNA prepared from the cells of strain C14 (Table 1), and the appearance of Trp+ transformants was checked. The transformation experiment for selection of Trp+ was carried out by a similar procedure, as described in the text.

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TABLE 6. Transduction and transformation mapping of the spo mutation in strain 5221a Unselected marker' No. of No. of Rif 1 1

Spo 1

Abs 1

transductants

transformants

187 64 0 10 24 0 0 14 112 "Transduction with phage PBS1 and transformation were carried out as described in the text. The donor strain was 5221 (cys' tip rifr spo abs), and the recipient strain was C14 (cysA trp+ rif spo+ abs+). Cys+ (Trp+) transductants or transformants were selected. b Donor and recipient markers are indicated by 1 and 0, respectively. Rif, Resistance to rifampin; Spo, sporulation; Abs, production of antibiotics. None of the other classes (110, 101, 011, 010, and 001) was found among the 211 transductants or 200 transformant clones. 0 0

tion, spo-4, that mapped between cysA and rif (data not presented). Further genetic and biochemical analyses of this mutant in correlation with similar mutants described by others (9, 10, 26) are under way. DISCUSSION From the results presented we propose that there is a close correlation between early sporulation events and establishment of lysogeny by temperate phages in vegetative cells of B. subtilis. Hong et al. (12) reported that S. typhimurium mutants defective in adenyl cyclase (cya gene) or in cyclic AMP receptor protein (crp gene) and some rifampin-resistant mutants are lysogenized at reduced frequency by phage P22. They postulated that in the wild-type bacteria the cyclic AMP receptor protein, in combination with cyclic AMP, activated bacterial RNA polymerase to transcribe certain phage genes required for the establishment of lysogeny and that, under the conditions of strong catabolite repression, the phage's decision between lysogeny and lysis was shifted to lysis. Similarly, Grodzicker et al. (7) reported the reduced ability to establish lysogeny by lambdoid phages in mutants of Escherichia coli. It has been suggested by various workers that bacterial sporulation is regulated, at least in part, by catabolite repression (6, 30, 32). Although neither cyclic AMP nor adenyl cyclase and phosphodiesterase activities have been detected in B. subtilis (13, 33), it seems worthwhile to study further the mechanism of the establishment of lysogeny and its correlation with early events of sporulation. From this line of investigation we hope that new information concerning the mechanism of initiation of sporulation will be revealed.

J. BACTERIOL.

Similar results were obtained with both 4105 and SPO2 (Table 2), although the latter showed a higher frequency of lysogenization in the wildtype strain. This suggests that the establishment of lysogeny by 4105 and SPO2 in B. subtilis is regulated by a common system. When 480 and A (Ah8O) infected some mutants of E. coli, they lysogenized similarly with reduced frequencies (7). Early-blocked asporogenous (Spo- AbsCpt-) mutants other than those that had spo mutations near the pheA-lys region were isolated. We found that their mutation sites were located near rif and that they were lysogenized by 4105 at a reduced frequency. spo-5 is closely linked to std and may be a mutation of the structural gene of RNA polymerase. Fine genetic analyses, as well as biochemical studies, are now being carried out. Sonenshein et al. (34) isolated and characterized a number of Rif' Spo- and Stdr Spo- mutants. Haworth and Brown reported (8) that mutations (ts) responsible for the temperature sensitivity of RNA polymerase mapped near the right side (opposite cysA) of rif (or std). Although the relationship between these mutations (spo and ts) and spo-5 is not certain, spo-5 is considered to be nearer to rif than to ts (8), based on the cotransfonnation frequencies with the cysA marker. Osburne and Sonenshein (25) also reported the behavior of phage 4105 in sporulating cells. When 4105 infected sporulating cells at early times (T, to T3), phage DNA entered the developing spores in a heat-stable forn, which might represent integration of the phage DNA into the host chromosome. Phage DNA in carrier spores produced by infection at later times (T4 to T6) was heat sensitive. Further investigations may be required to decide whether this heat-stable form of phage DNA represents the lysogenized state. There is no difference between the frequency of lysogenization of the wild-type strain by 4105 in the sporulating phase (T1 to T2) and that in the vegetative phase (details to be published elsewhere). ACKNOWLEDGMENTIS We are grateful to S. Okubo, J. Spizizen, P. Schaeffer, A. Uchida, and H. Saito for providing us with the bacterial and phage strains used in this study and to G. B. Whitfield, Jr., of the Upjohn Company for the gift of streptolydigin. This investigation was supported by grants from the Ministry of Education, Science and Culture of Japan.

LITERATURE CITED 1. Anagnostopoulos, C., and J. Spizizen. 1961. Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81:741-746. 2. BirdseU, D. C., G. M. Hathaway, and L Rutberg. 1969. Characterization of temperate Bacillus bacterio-

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phage 4105. J. Virol. 4:264-270. 3. Brehm, S. P., F. LeHegarat, and J. A. Hoch. 1975. Deox.yribonucleic acid-binding proteins in vegetative Bacillus subtilis: alteration caused by stage 0 sporulation mutants. J. Bacteriol. 124:977-984. 4. Brehm, S. P., P. S. Stephen, and J. A. Hoch. 1973. Phenotypes of pleiotropic-negative sporulation mutants of Bacillus subtilis. J. Bacteriol. 115:1063-1070. 5. Brevet, J. 1976. Evolution of the transcription complex during sporulation of Bacillus subtilis. J. Bacteriol. 125:74-83. 6. Freese, E., Y. K. Oh, E. B. Freese, M. D. Diesterhaft, and C. Prasad. 1972. Suppression of sporulation of Bacillus subtilis, p. 212-221. In H. 0. Halvorson, R. Hansor., and L. L. Campbell (ed.), Spores V. American Society for Microbiology, Washington, D.C. 7. Grodzicker, T., R. R. Arditti, and H. Eisen. 1972. Establishment of repression by lambdoid phage in catabolite activator protein and adenyl cyclase mutants of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 69:366-370. 8. Haworth, S. R., and L. R. Brown. 1973. Genetic analysis of ribonucleic acid polymerase mutants of Bacillus subtilis. J. Bacteriol. 114:103-113. 9. Hoch, J. A. 1976. Genetics of bacterial sporulation. Adv. Genet. 18:69-99. 10. Hoch, J. A., and J. L. Mathews. 1973. Chromosomal location of pleiotropic negative sporulation mutations in Bacillus subtilis. Genetics 73:215-228. 11. Hoch, J. A., and J. Spizizen. 1969. Genetic control of some early events in sporulation in Bacillus subtilis 168, p. 112-120. In L. L. Champbell (ed.), Spores IV. American Society for Microbiology, Bethesda, Md. 12. Hong, J.-S., G. R. Smith, and B. N. Ames. 1971. Adenosine 3':5'-cyclic monophosphate concentration in the bacterial host regulates the viral decision between lysogeny and lysis. Proc. Natl. Acad. Sci. U.S.A. 68:2258-2262. 13. Ide, M. 1971. Adenyl cyclase of bacteria. Arch. Biochem. Biophys. 144:262-268. 14. Inselberg, J. W., T. Eremenko-Volpe, L. Greenwald, W. L. Meadow, and J. Marmur. 1969. Physical and genetic mapping of the SP02 prophage on the chromosome of Bacillus subtilis 168. J. Virol. 3:627-628. 15. Ionesco, M., J. Michel, B. Cami, and P. Schaeffer. 1970. Genetics of sporulation in Bacillus subtilis Marburg. J. Appl. Bacteriol. 33:13-24. 16. Ito, J., and J. Spizizen. 1971. Abortive infection of sporulating Bacillus subtilis 168 by 42 bacteriophage. J. Virol. 7:515-523. 17. Korch, C. T., and R. H. Doi. 1971. Electron microscopy of the altered spore morphology of a ribonucleic acid polymerase mutant of Bacillus subtilis. J. Bacteriol.

105:1110-1118. 18. Lennox, C. S. 1955. Transduction of linked genetic characters of the host by bacteriophage P1. Virology

1:190-206. 19. Linn, T., and R. Losick. 1976. The program of protein synthesis during sporulation in Bacillus subtilis. Cell 8:103-114. 20. Linn, T. G., A. L. Greenleaf, R. G. Shorenstein, and R. Losick. 1973. Loss of the sigma activity of RNA polymerase of Bacillus subtilis during sporulation. Proc. Natl. Acad. Sci. U.S.A. 70:1865-1869. 21. Losick, R., R. G. Shorenstein, and A. L Sonenshein. 1970. Structural alteration of RNA polymerase during sporulation. Nature (London) 227:910-913. 22. Mukherjee, P. K., and H. Paulus. 1977. Biological function of gramicidin: studies of gramicidin-negative mu-

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